The bacterial toxins, cholera toxin and pertussis toxin, are responsible in part for the pathogenesis of cholera and pertussis, respectively. The toxins exert their effects on cells through the ADP-ribosylation of guanine nucleotide-binding (G) proteins that are critical for signaling from receptors to their intracellular targets (e.g., adenylyl cyclase). ADP-ribosyltransferases similar to the bacterial toxins are present in animal cells. Also present are ADP-ribosylarginine hydrolases, enzymes that cleave the ADP-ribose-protein linkage. It would thus appear that, in animal cells, an ADP-ribosylation cycle exists which catalyzes the reversible modification of proteins. Hydrolase activity was observed 'in a variety of animal species, with significantly higher levels in rat and mouse than in guinea pig and calf. In rat tissues, specific activity was higher in brain, spleen and testis than in lung, heart, liver and skeletal muscle. The rat brain hydrolase was purified approximately 20,000-fold and exhibited one major band on sodium dodecyl sulfate-polyacrylamide gels consistent with a protein of approximately 39 kDa. Rabbit anti-rat hydrolase polyclonal antibodies recognized proteins of approximately 30 kDa in partially purified hydrolase preparations from rat, mouse, and calf brains and turkey erythrocytes. Based on amino acid sequence obtained from the purified protein, oligonucleotide and polymerase chain reaction (PCR)-generated cDNA probes were synthesized and used to screen a rat brain Lambda ZAP library. Inserts from two independent clones yielded a composite open reading frame of 1086 bp. The hydrolase cDNA was expressed in E. coli as a fusion protein that exhibited a Mg2+- and dithiothreitol- dependent activity similar to that of the rat brain enzyme. A PCR-generated coding region cDNA hybridized to a 1.7 kb mRNA on Northern analysis of poly(A)+ RNA from rat and mouse but not human, bovine, or rabbit tissues and cells. The data are compatible with partial conservation of hydrolase structure across animal species.